星孢菌素对山羊颞下颌关节盘细胞自组装基体收缩的影响

doi:10.7518/hxkq.2019.03.009

华西口腔医学杂志 ›› 2019, Vol. 37 ›› Issue (3): 275-279.doi: 10.7518/hxkq.2019.03.009

星孢菌素对山羊颞下颌关节盘细胞自组装基体收缩的影响

保善英¹,包广洁^2,³,唐玉尧¹,刘琳^2,³,康宏¹()

1.兰州大学口腔医学研究所,兰州 730000
2.西北民族大学口腔医学国家民委重点实验室
3.西北民族大学甘肃省口腔疾病研究重点实验室,兰州 730030

收稿日期:2018-03-05 修回日期:2019-03-16 出版日期:2019-06-01 发布日期:2019-06-12
通讯作者: 康宏 E-mail:kanghong@lzu.edu.cn
作者简介:保善英,住院医师,硕士,E-mail:baoshy15@lzu.edu.cn
基金资助:
国家自然科学基金(81660189);西北民族大学中央高校基本科研业务费(2yp2015014);西北民族大学中央高校基本科研业务费(31920170167)

Effects of staurosporine on the contraction of self-assembled constructs of goat temporomandibular joint disc cells

Shanying Bao¹,Guangjie Bao^2,³,Yuyao Tang¹,Lin Liu^2,³,Hong Kang¹()

1.Institute of Stomatology, Lanzhou University, Lanzhou 730000, China
2.Key Laboratory of Stomatology of State Ethnic Affairs Commission, Northwest Minzu University, Lanzhou 730030, China
3.Key Laboratory of Oral Diseases of Gansu Province, Northwest Minzu University, Lanzhou 730030, China

Received:2018-03-05 Revised:2019-03-16 Online:2019-06-01 Published:2019-06-12
Contact: Hong Kang E-mail:kanghong@lzu.edu.cn
Supported by:
The National Natural Science Foundation of China(81660189);Fundamental Scientific Research Funding of Central University for Northwest Minzu University(2yp2015014);Fundamental Scientific Research Funding of Central University for Northwest Minzu University(31920170167)

摘要/Abstract

摘要：

目的探索不同浓度星孢菌素对山羊颞下颌关节盘细胞自组装基体收缩性及细胞外基质合成的影响。方法单层培养山羊颞下颌关节盘细胞至P3代,将5.5×10 ⁶个细胞在不同浓度（0、0.1、1、10、100 nmol·L ^-1）星孢菌素的完全培养基中分别接种于三维柱状琼脂糖井;每天每井换液,培养1周后观察自组装基体的形态变化;冰冻切片染色显示自组装基体细胞外基质及α-平滑肌动蛋白（α-SMA）的分布;酶联免疫吸附测定（ELISA）和Blyscan试剂盒检测基体细胞外基质含量的变化。结果山羊颞下颌关节盘细胞接种4 h后,星孢菌素组和对照组细胞均在琼脂糖井内自组装,形成一圆盘状基体,之后变成球形。各组自组装基体在1周时天狼星红染色可见基体内大量胶原纤维分布;番红染色显示基体内纤维软骨样细胞明显分泌糖胺多糖（GAGs）;Ⅰ型胶原（ColⅠ）在免疫组织化学染色中呈强阳性;α-SMA免疫组织化学染色为弱阳性。星孢菌素组自组装基体内ColⅠ和GAGs较高,其中10 nmol·L ^-1组最明显（P<0.01）。结论星孢菌素对自组装基体的收缩有一定干预效果,但不是非常突出;它更有利于细胞外基质的合成。

关键词: 星孢菌素, 自组装基体, 颞下颌关节盘

Abstract:

Objective The effects of the staurosporine on contraction of self-assembled constructs and extracellular matrix syntheses of goat temporomandibular joint discs were investigated. Methods Goat temporomandibular joint disc cells were isolated and cultured to P3, and 5.5×10 ⁶cells were combined with different concentrations of staurosporine (0, 0.1, 1, 10, 100 nmol·L ^-1) in agarose wells and cultured for one week. The samples were frozen and sectioned. Safranin-O, Picro-sirius red and immunohistochemical staining were performed to observe the distributions of the extracellular matrix and the expression of alpha-smooth muscle actin (α-SMA). Enzyme linked immunosorbent assay (ELISA) and Blyscan kits were utilized to quantitatively detect the contents of type Ⅰ collagen (ColⅠ) and glycosaminoglycans (GAGs). Results Each group of goat temporomandibular joint disc cells in the agarose wells were gathered to self-assemble into a disc-shaped base for 4 hours and then to gradually contract into a round shape. The Picro-sirius red staining was strong and indicated collagen distribution. The Safranin-O staining observed GAGs throughout the entire construct. The expression of ColⅠ was strongly positive in the staurosporine groups; however, the expression of α-SMA was weak. ColⅠ and GAGs contents in the staurosporine groups were greater than that of the control group, especially in the 10 nmol·L ^-1 group (P<0.01). Conclusion Staurosporine has a certain effect on the shrinkage of self-assembled constructs; however, such effect is not prominent. Staurosporine contributes to the construction synthesis of extracellular matrix.

Key words: staurosporine, self-assembled construct, temporomandibular joint disc

中图分类号:

R782.6

保善英,包广洁,唐玉尧,刘琳,康宏. 星孢菌素对山羊颞下颌关节盘细胞自组装基体收缩的影响[J]. 华西口腔医学杂志, 2019, 37(3): 275-279.

Shanying Bao,Guangjie Bao,Yuyao Tang,Lin Liu,Hong Kang. Effects of staurosporine on the contraction of self-assembled constructs of goat temporomandibular joint disc cells[J]. West China Journal of Stomatology, 2019, 37(3): 275-279.

图/表 3

参考文献 18

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组别	直径/mm	湿重/mg	ColⅠ/mg	GAGs/μg
对照组	1.79±0.040 4	1.66±0.043 7	0.041±0.001 4	5.378±0.222
0.1 nmol·L^-1星孢菌素组	2.00±0.036 7	1.86±0.041 9	0.051±0.001 5	5.832±0.202
1 nmol·L^-1星孢菌素组	2.30±0.027 8	2.17±0.049 1	0.059±0.001 6	6.377±0.178
10 nmol·L^-1星孢菌素组	3.03±0.032 4*	2.48±0.041 3*	0.078±0.001 5*	8.283±0.255*
100 nmol·L^-1星孢菌素组	2.61±0.048 4	2.29±0.034 0	0.068±0.001 9	6.922±0.212

星孢菌素对山羊颞下颌关节盘细胞自组装基体收缩的影响

Effects of staurosporine on the contraction of self-assembled constructs of goat temporomandibular joint disc cells

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